Modification of the Antibiotic, Colistin, with Dextrin Causes Enhanced Cytotoxicity and Triggers Apoptosis in Myeloid Leukemia.

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.

Evaluation of the ecological risk of pesticide residues from the European LUCAS Soil monitoring 2018 survey.

The 2018 LUCAS (Land Use and Coverage Area frame Survey) Soil Pesticides survey provides a European Union (EU)-scale assessment of 118 pesticide residues in more than 3473 soil sites. This study responds to the policy need to develop risk-based indicators for pesticides in the environment. Two mixture risk indicators are presented for soil based, respectively, on the lowest and the median of available No Observed Effect Concentration (NOECsoil,min and NOECsoil,50) from publicly available toxicity datasets. Two further indicators were developed based on the corresponding equilibrium concentration in the aqueous phase and aquatic toxicity data, which are available as species sensitivity distributions. Pesticides were quantified in 74.5% of the sites. The mixture risk indicator based on the NOECsoil,min exceeds 1 in 14% of the sites and 0.1 in 23%. The insecticides imidacloprid and chlorpyrifos and the fungicide epoxiconazole are the largest contributors to the overall risk. At each site, one or a few substances drive mixture risk. Modes of actions most likely associated with mixture effects include modulation of acetylcholine metabolism (neonicotinoids and organophosphate substances) and sterol biosynthesis inhibition (triazole fungicides). Several pesticides driving the risk have been phased out since 2018. Following LUCAS surveys will determine the effectiveness of substance-specific risk management and the overall progress toward risk reduction targets established by EU and UN policies. Newly generated data and knowledge will stimulate needed future research on pesticides, soil health, and biodiversity protection. Integr Environ Assess Manag 2024;00:1-15. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Quantitative analysis of the compliance of EU Sewage Sludge Directive by using the heavy metal concentrations from LUCAS topsoil database.

In the European Union (EU), a common understanding of the potential harmful effect of sewage sludge (SS) on the environment is regulated by the Sewage Sludge Directive 86/278/EEC (SSD). Limit values (LVs) for concentrations of heavy metals in soil are listed in Impact Assessment of this directive, and they were transposed by EU member states using different criteria. Member states adopted either single limit values or based on soil factors such as pH and texture to define the maximum limit values for concentrations of heavy metals in soils. Our work presents the first quantitative analysis of the SSD at the European level by using the Land Use and Coverage Area Frame Survey (LUCAS) 2009 topsoil database. The reference values at the European level were arranged taking into account the upper value (EU_UL) and the lower value (EU_LL) for each heavy metal (arsenic, cadmium, copper, chromium, mercury, nickel, lead, and zinc) as well as taking into account the pH of the soil (cadmium, copper, mercury, nickel, lead, and zinc) as introduced in the SSD Annex IA. Single and integrated contamination rate indices were developed to identify those agricultural soils that exceeded the reference values for each heavy metal. In total, 10%, 36%, and 19% of the LUCAS 2009 topsoil samples exceeded the limit values. Additionally, 12% and 16% of agricultural soils exceeded the concentration of at least one single heavy metal when European LVs were fixed following the soil pH in Strategy II compared to those national ones in Strategy I. Generally, all member states apply similar or stricter limit values than those laid down in the SSD. Our work indicates that choosing LVs quantitatively affects further actions such as monitoring and remediation of contaminated soils. The actual soil parameters, such as heavy metal concentrations and soil pH values from the LUCAS 2009 topsoil database, could be used by SSD-involved policy stakeholders not only to lay down the LVs for concentrations of heavy metal in soils but also for monitoring the SSD compliance grade by using the LUCAS surveys over time (past and upcoming LUCAS datasets).

Interaction effects of pH and land cover on soil microbial diversity are climate-dependent.

Factors regulating the diversity and composition of soil microbial communities include soil properties, land cover and climate. How these factors interact at large scale remains poorly investigated. Here, we used an extensive dataset including 715 locations from 24 European countries to investigate the interactive effects of climatic region, land cover and pH on soil bacteria and fungi. We found that differences in microbial diversity and community composition between land cover types depended on the climatic region. In Atlantic, Boreal and Continental regions, microbial richness was higher in croplands and grasslands than woodlands while richness in Mediterranean areas did not vary significantly among land cover types. These differences were further related to soil pH, as a driver of bacterial and fungal richness in most climatic regions, but the interaction of pH with land cover depended on the region. Microbial community composition differed the most between croplands and woodlands in all regions, mainly due to differences in pH. In the Mediterranean region, bacterial communities in woodlands and grasslands were the most similar, whereas in other regions, grassland and cropland-associated bacteria showed more similarity. Overall, we showed that key factors interact in shaping soil microbial communities in a climate-dependent way at large scale.

Soil organic carbon stocks in European croplands and grasslands: How much have we lost in the past decade?

The EU Soil Strategy 2030 aims to increase soil organic carbon (SOC) in agricultural land to enhance soil health and support biodiversity as well as to offset greenhouse gas emissions through soil carbon sequestration. Therefore, the quantification of current SOC stocks and the spatial identification of the main drivers of SOC changes is paramount in the preparation of agricultural policies aimed at enhancing the resilience of agricultural systems in the EU. In this context, changes of SOC stocks (Δ SOCs) for the EU + UK between 2009 and 2018 were estimated by fitting a quantile generalized additive model (qGAM) on data obtained from the revisited points of the Land Use/Land Cover Area Frame Survey (LUCAS) performed in 2009, 2015 and 2018. The analysis of the partial effects derived from the fitted qGAM model shows that land use and land use change observed in the 2009, 2015 and 2018 LUCAS campaigns (i.e. continuous grassland [GGG] or cropland [CCC], conversion grassland to cropland (GGC or GCC) and vice versa [CGG or CCG]) was one of the main drivers of SOC changes. The CCC was the factor that contributed to the lowest negative change on Δ SOC with an estimated partial effect of -0.04 ± 0.01 g C kg-1 year-1 , while the GGG the highest positive change with an estimated partial effect of 0.49 ± 0.02 g C kg-1 year-1 . This confirms the C sequestration potential of converting cropland to grassland. However, it is important to consider that local soil and environmental conditions may either diminish or enhance the grassland's positive effect on soil C storage. In the EU + UK, the estimated current (2018) topsoil (0-20 cm) SOC stock in agricultural land below 1000 m a.s.l was 9.3 Gt, with a Δ SOC of -0.75% in the period 2009-2018. The highest estimated SOC losses were concentrated in central-northern countries, while marginal losses were observed in the southeast.

Cadmium in topsoils of the European Union - An analysis based on LUCAS topsoil database.

Cadmium (Cd) is a naturally occurring element that can accumulate in the soil through the application of fertilisers containing cadmium and as a waste of industrial processes. Cadmium inputs in the soil have increased significantly (+50 %) during the 20th century as a result of the application of fertilisers and sewage sludge, and also due to local contamination (e.g. waste dumping, mining) and industrial emissions (e.g. zinc smelters). Using the 21,682 soil samples from the LUCAS soil survey, we aim to estimate the spatial distribution of the concentration of Cd in the European Union (EU) and UK topsoil. Out of the total, 72.6 % of the samples have Cd values <0.07 mg kg-1, 21.6 % in the range 0.07-1 mg kg-1 and the remaining 5.5 % higher than the threshold of 1 mg kg-1, which is generally considered the limit for risk assessment. The mean Cd value in the EU topsoils is 0.20 mg kg-1, slightly higher in grasslands (0.24 mg kg-1) compared to croplands (0.17 mg kg-1). Applying an ensemble of machine learning models supported by a variety of environmental descriptors, we created maps of Cd distribution at a resolution of 100 m. The ensemble approach included five models and increased the prediction accuracy to R2 of 0.45 (an increase of 0.1 compared to best single model performance). The approach used resulted in a high predictive power for the general Cd distribution, while also identifying hotspots of Cd contamination. Natural factors influencing Cd levels include soil properties (pH, clay), topography, soil erosion, and leaching. As anthropogenic factors, we identified phosphorus inputs to agricultural lands as the most important for Cd levels. The application of the EU Fertiliser Directive should further limit Cd inputs and potentially the Cd content in soils.

Ecosystem type drives soil eukaryotic diversity and composition in Europe.

Soil eukaryotes play a crucial role in maintaining ecosystem functions and services, yet the factors driving their diversity and distribution remain poorly understood. While many studies focus on some eukaryotic groups (mostly fungi), they are limited in their spatial scale. Here, we analyzed an unprecedented amount of observational data of soil eukaryomes at continental scale (787 sites across Europe) to gain further insights into the impact of a wide range of environmental conditions (climatic and edaphic) on their community composition and structure. We found that the diversity of fungi, protists, rotifers, tardigrades, nematodes, arthropods, and annelids was predominantly shaped by ecosystem type (annual and permanent croplands, managed and unmanaged grasslands, coniferous and broadleaved woodlands), and higher diversity of fungi, protists, nematodes, arthropods, and annelids was observed in croplands than in less intensively managed systems, such as coniferous and broadleaved woodlands. Also in croplands, we found more specialized eukaryotes, while the composition between croplands was more homogeneous compared to the composition of other ecosystems. The observed high proportion of overlapping taxa between ecosystems also indicates that DNA has accumulated from previous land uses, hence mimicking the land transformations occurring in Europe in the last decades. This strong ecosystem-type influence was linked to soil properties, and particularly, soil pH was driving the richness of fungi, rotifers, and annelids, while plant-available phosphorus drove the richness of protists, tardigrades, and nematodes. Furthermore, the soil organic carbon to total nitrogen ratio crucially explained the richness of fungi, protists, nematodes, and arthropods, possibly linked to decades of agricultural inputs. Our results highlighted the importance of long-term environmental variables rather than variables measured at the time of the sampling in shaping soil eukaryotic communities, which reinforces the need to include those variables in addition to ecosystem type in future monitoring programs and conservation efforts.

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles.

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid-based therapeutics transported by nanoparticles. Utilizing a siRNA-targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3-LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID). A cell-based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data-rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid-phase endocytosis as a productive cellular entry route. With this new knowledge, MC3-LNP is re-engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE-ID approach can be broadly applicable for optimizing nanomedicine-based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid-based therapeutics.

Stereoisomer-Dependent Membrane Association and Capacity for Insulin Delivery Facilitated by Penetratin.

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Patterns in soil microbial diversity across Europe.

Factors driving microbial community composition and diversity are well established but the relationship with microbial functioning is poorly understood, especially at large scales. We analysed microbial biodiversity metrics and distribution of potential functional groups along a gradient of increasing land-use perturbation, detecting over 79,000 bacterial and 25,000 fungal OTUs in 715 sites across 24 European countries. We found the lowest bacterial and fungal diversity in less-disturbed environments (woodlands) compared to grasslands and highly-disturbed environments (croplands). Highly-disturbed environments contain significantly more bacterial chemoheterotrophs, harbour a higher proportion of fungal plant pathogens and saprotrophs, and have less beneficial fungal plant symbionts compared to woodlands and extensively-managed grasslands. Spatial patterns of microbial communities and predicted functions are best explained when interactions among the major determinants (vegetation cover, climate, soil properties) are considered. We propose guidelines for environmental policy actions and argue that taxonomical and functional diversity should be considered simultaneously for monitoring purposes.

PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.

Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.

Influence of Folate-Targeted Gold Nanoparticles on Subcellular Localization and Distribution into Lysosomes.

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0-100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process.

Nanoparticle entry into cells; the cell biology weak link.

Nanoparticles (NP) are attractive options for the therapeutic delivery of active pharmaceutical drugs, proteins and nucleic acids into cells, tissues and organs. Research into the development and application of NP most often starts with a diverse group of scientists, including chemists, bioengineers and material and pharmaceutical scientists, who design, fabricate and characterize NP in vitro (Stage 1). The next step (Stage 2) generally investigates cell toxicity as well as the processes by which NP bind, are internalized and deliver their cargo to appropriate model tissue culture cells. Subsequently, in Stage 3, selected NP are tested in animal systems, mostly mouse. Whereas the chemistry-based development and analysis in Stage 1 is increasingly sophisticated, the investigations in Stage 2 are not what could be regarded as 'state-of-the-art' for the cell biology field and the quality of research into NP interactions with cells is often sub-standard. In this review we describe our current understanding of the mechanisms by which particles gain entry into mammalian cells via endocytosis. We summarize the most important areas for concern, highlight some of the most common mis-conceptions, and identify areas where NP scientists could engage with trained cell biologists. Our survey of the different mechanisms of uptake into cells makes us suspect that claims for roles for caveolae, as well as macropinocytosis, in NP uptake into cells have been exaggerated, whereas phagocytosis has been under-appreciated.

Challenges of and opportunities for protecting European soil biodiversity.

Soil biodiversity and related ecosystem functions are neglected in most biodiversity assessments and nature conservation actions. We examined how society, and particularly policy makers, have addressed these factors worldwide with a focus on Europe and explored the role of soils in nature conservation in Germany as an example. We reviewed past and current global and European policies, compared soil ecosystem functioning in- and outside protected areas, and examined the role of soils in nature conservation management via text analyses. Protection and conservation of soil biodiversity and soil ecosystem functioning have been insufficient. Soil-related policies are unenforceable and lack soil biodiversity conservation goals, focusing instead on other environmental objectives. We found no evidence of positive effects of current nature conservation measures in multiple soil ecosystem functions in Europe. In German conservation management, soils are considered only from a limited perspective (e.g., as physicochemical part of the environment and as habitat for aboveground organisms). By exploring policy, evidence, and management as it relates to soil ecosystems, we suggest an integrative perspective to move nature conservation toward targeting soil ecosystems directly (e.g., by setting baselines, monitoring soil threats, and establishing a soil indicator system).

La biodiversidad del suelo y las funciones ambientales relacionadas se dejan de lado en la mayoría de las evaluaciones de la biodiversidad y de las acciones de conservación de la naturaleza. Analizamos cómo la sociedad, y particularmente los formuladores de políticas, han abordado estos factores a nivel mundial con un enfoque en Europa y exploramos como ejemplo el papel de los suelos en la conservación de la naturaleza en Alemania. Revisamos las políticas mundiales y europeas en el pasado y en la actualidad, comparamos el funcionamiento ambiental del suelo dentro y fuera de las áreas protegidas y examinamos el papel de los suelos en la gestión de la conservación por medio del análisis de textos. La protección y la conservación de la biodiversidad y el funcionamiento ambiental del suelo han sido insuficientes. Las políticas relacionadas con el suelo son inaplicables y carecen de objetivos de conservación para su biodiversidad, pues se enfocan más bien en otros objetivos ambientales. No descubrimos evidencias de los efectos positivos de las medidas actuales de conservación en múltiples funciones ambientales del suelo en Europa. En la gestión alemana de la conservación, los suelos sólo se consideran desde una perspectiva limitada (p. ej.: como una parte físico química del ambiente y como hábitat para los organismos que habitan por encima de él). Mediante la exploración de la política, evidencias y gestión conforme se relaciona con los ecosistemas del suelo, sugerimos una perspectiva integrada para dirigir a la conservación hacia el enfoque directo sobre los ecosistemas del suelo (p. ej.: al establecer líneas base, monitorear las amenazas para el suelo y establecer un sistema indicador del suelo).

Highly-fluorescent BODIPY-functionalised metallacages as drug delivery systems: synthesis, characterisation and cellular accumulation studies.

With the aim of designing new metallosupramolecular architectures for drug delivery, research has focused on porous 3-dimensional (3D)-metallacages able to encapsulate cytotoxic agents protecting them from metabolism while targeting them to cancer sites. Here, two self-assembled [Pd2L4]4+ cages (CG1 and CG2) featuring 3,5-bis(3-ethynylpyridine)phenyl ligands (L) exo-functionalised with dipyrromethene (BODIPY) groups have been synthesised and characterised by different methods, including NMR spectroscopy and mass spectrometry. 1H NMR spectroscopy studies shows that the cages are able to encapsulate the anticancer drug cisplatin in their hydrophobic cavity, as evidenced by electrostatic potential (ESP) analysis based on XRD studies. The stability of the cages in an aqueous environment, and in the presence of the intracellular reducing agent glutathione, has been confirmed by UV-visible absorption spectroscopy. The luminescence properties of the cages enabled the investigation of their cellular uptake and intracellular localisation in human cancer cells by confocal laser scanning microscopy. In melanoma A375 cells, cage CG1 is taken up via active transport and endocytic trafficking studies show little evidence of transport through the early endosome while the cages accumulated in melanosomes rather than lysosomes. The antiproliferative activity of the lead cage was investigated in A375 together with two breast cancer cell lines, SK-BR-3 and MCF7. While the cage per se is non-cytotoxic, very different antiproliferative effects with respect to free cisplatin were evidenced for the [(cisplatin)2⊂CG1·BF4] complex in the various cell lines, which correlate with its different intracellular localisation profiles. The obtained preliminary results provide a new hypothesis on how the subcellular localisation of the cage affects the cisplatin intracellular release.

Fluid-Phase Endocytosis and Lysosomal Degradation of Bovine Lactoferrin in Lung Cells.

The iron-binding protein lactoferrin and the cell-penetrating peptides derived from its sequence utilise endocytosis to enter different cell types. The full-length protein has been extensively investigated as a potential therapeutic against a range of pathogenic bacteria, fungi, and viruses, including SARS-CoV-2. As a respiratory antiviral agent, several activity mechanisms have been demonstrated for lactoferrin, at the extracellular and plasma membrane levels, but as a protein that enters cells it may also have intracellular antiviral activity. Characterisation of lactoferrin's binding, endocytic traffic to lysosomes, or recycling endosomes for exocytosis is lacking, especially in lung cell models. Here, we use confocal microscopy, flow cytometry, and degradation assays to evaluate binding, internalisation, endocytic trafficking, and the intracellular fate of bovine lactoferrin in human lung A549 cells. In comparative studies with endocytic probes transferrin and dextran, we show that lactoferrin binds to negative charges on the cell surface and actively enters cells via fluid-phase endocytosis, in a receptor-independent manner. Once inside the cell, we show that it is trafficked to lysosomes where it undergoes degradation within two hours. These findings provide opportunities for investigating both lactoferrin and derived cell-penetrating peptides activities of targeting intracellular pathogens.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.